Our studies address a critical problem in hemoglobin (Hb) biology: how inherently unstable globin protein subunits are folded and maintained during normal and pathological erythropoiesis. We discovered alpha hemoglobin stabilizing protein (AHSP), an erythroid protein that specifically binds free alpha globin subunit, stabilizes its structure and limits its pro-oxidant activities. Our preliminary studies suggest two distinct functions for AHSP. First, to detoxify excess alpha globin that accumulates during normal erythropoiesis and in various anemias, particularly beta thalassemia. Second, to fold and stabilize newly formed alpha globin subunits en route to HbA (alpha beta) synthesis. Of potential importance to both functions, we discovered that degradation of AHSP mRNA is accelerated by iron, an essential component of HbA and determinant of nascent globin protein stability. Our overall view is that AHSP facilitates normal HbA synthesis and also buffers against imbalances that arise from genetic or environmental stresses, such as thalassemias and iron deficiency. Now we seek to better understand AHSP activities and their relevance to human health. Aim 1 uses mouse genetics to investigate AHSP functions in vivo. We will examine the consequences of manipulated AHSP expression in thalassemias and create Ahsp gene missense mutations in mice to probe mechanisms of AHSP protein function. Aim 2 studies the biochemical properties of AHSP. We will test in vitro if AHSP promotes reconstitution of HbA from its purified apo-globin and heme components and search for new erythroid proteins that interact with alpha globin-AHSP complexes. Aim 3 examines the mechanisms by which iron regulates AHSP expression and the physiological implications of this pathway during altered iron homeostasis. If successful, our work will establish new basic principles of Hb biology and erythropoiesis. In addition, there are potential practical long-term benefits. For example, understanding how AHSP detoxifies excess alpha Hb should illustrate novel therapeutic approaches for human beta thalassemias. Elucidating the role of AHSP in HbA synthesis may provide tools to optimize the manufacture of recombinant Hb-based blood substitutes. Finally, defining functional interactions between iron and AHSP could provide insights into the pathophysiology and management of iron overload and deficiency states.